The temporary interruption of blood flow to tissue is a necessary step in many surgical procedures, such as cardiac surgery and organ preservation or transplantation, in order to prevent blood loss and to facilitate surgery. Blood vessels can also become blocked during disease events, such as myocardial infarction, thrombotic stroke, embolic vascular occlusions, angina pectoris, and peripheral vascular insufficiency. The lack of blood supply under these circumstances results in ischemia, which is reversed upon reperfusion of the ischemic tissue with blood or another oxygen-carrying solution. While this readmission of oxygen is critical for the continued function of the tissue, it is generally accepted that the newly introduced oxygen contributes to the formation of oxygen-derived free radicals that cause tissue damage. One mechanism by which introduced oxygen is made toxic is by conversion to superoxide by xanthine oxidase. Levels of this enzyme can become increased during the ischemic period. Simultaneously, levels of reducing, detoxifying agents, such as glutathione, are depleted. Tissue damage occuring as a result of these events is known as reperfusion injury, and is known to occur during reperfusion with blood and is anticipated under some circumstances with the use of blood substitutes.
One class of blood substitutes, the hemoglobin-based oxygen carriers (HBOCs), are comprised of chemically modified acellular hemoglobin. Acellular hemoglobin presents an additional source of potentially damaging reactive oxygen species. Hemoglobin in blood is normally contained within the red blood cells of the blood, in which it circulates through the body to fulfil its oxygen-transporting function. Hemoglobin in the red cells binds oxygen as the blood circulates through the lungs, delivers the oxygen to the body tissues and releases it there, for normal metabolic functions. The chemical behavior of hemoglobin in blood is constrained by its presence in the red cells, which also contain many other components such as enzymes which influence the chemical behavior of hemoglobin therein. When hemoglobin is extracted from red cells and purified ready for use as an acellular oxygen-transporter in blood substitute applications, the chemical influence on the hemoglobin of the other red cell components, and vice versa, is lost.
One of these influences relates to oxygen-hemoglobin reactions, and the generation of toxic oxygen species. Oxidation of hemoglobin by liganded oxygen produces met-hemoglobin, in which heme iron is oxidized to the Fe (III) state, and in which the oxygen free radical “superoxide”, O2— is generated. Met-hemoglobin does not have any significant useful function, since it is incapable of binding and transporting oxygen. Superoxide is, however, linked to a number of deleterious effects in the body, such as oxidative damage and injury to vascular components including endothelium and sub-endothelial tissue. In the red blood cell, enzymes are present to convert these toxic oxygen species to harmless by-products. Thus, the met-hemoglobin reductase enzymatic system is present to reduce the met-hemoglobin to hemoglobin. Superoxide dismutase and catalase are present, respectively to convert superoxide to hydrogen peroxide, and to convert hydrogen peroxide to water and molecular oxygen.
Hemoglobin outside the red cell has no such enzymatic reagents at hand to deal with these oxidation by-products. Consequently, the use of acellular hemoglobin as an oxygen-transporter may produce excessive quantities of deleterious oxidation products such as superoxide, arising from oxygen bound or becoming bound to the hemoglobin itself. Likewise, oxygen dissolved in the HBOC solution, or in the admixed blood reintroduced to the ischemic tissue, may result in reactive oxygen species giving rise to reperfusion injury by the mechanisms described above. However, the need to regain oxygen supply is paramount, and overrides the risks associated with reperfusion injury and introduction of oxygen radicals.
It is an object of the present invention to provide a novel hemoglobin composition capable of providing oxygen while overcoming or at least diminishing the above problem.